Plate layout

Shows the layout of samples, controls (e.g., negative controls (NC), Calibrator controls (CAL) and special wells if any (e.g., AQ sample controls (AQSC)) on the 96 well assay plate.

Plate_01

Plate_01: XML_v1.3.0_with_AQ.xml
1 2 3 4 5 6 7 8 9 10 11 12
A X252L X268L X254L X270L X315L X331L X317L X333L X284L X300L X286L X302L
B X253L X269L X255L X271L X316L X332L X318L X334L X285L X301L X287L X303L
C X256L X272L X258L X274L X319L X335L X321L X336L X288L X304L X290L X306L
D X257L X273L X259L X275L X320L NC 3 X322L NC 1 X289L X305L X291L X307L
E X260L X276L X262L X278L X323L NC 4 X325L NC 2 X292L X308L X294L X310L
F X261L X277L X263L X279L X324L AQSC 2 X326L CAL 2 X293L X309L X295L X31AL
G X264L X280L X266L X282L X327L AQSC 3 X329L CAL 3 X296L X311L X298L X313L
H X265L X281L X267L X283L X328L AQSC 1 X330L CAL 1 X297L X312L X299L X314L

Read summary

Summary of the number and classification of reads for the assay run

  • Parseable - Valid next generation sequencing (NGS) read (assay signal or background)
  • Parseable Match - Valid NGS read (assay signal)
  • Parseable Non-match - Vaild NGS read (assay background)
  • Unparseable - Sequencing background
  • Total wells - Total number of samples + controls on plate
  • Total targets - Total number of assay targets (including internal controls)
  • # of zeros (%) - Number and percentage of targets * samples which had zero sequencing reads (no assay signal)

Internal controls (IC)

Each sample of the assay is spiked with the same concentration of at least one internal control (IC). This is used both as part of a well normalization procedure and as a method for assessing the uniformity of the assay run. SD: standard deviation. CV: coefficient of variation

Internal Control (IC) summary - mCherry

Calibrator controls (CAL)

CAL samples are pooled plasma controls used both to normalize samples between experiments and to assess performance (e.g., precision) of the assay.

Note: %CV in this table does not exclude values below LOD. See Coefficient of variation section for %CV (before and after normalization) that excludes values below LOD.

IPC summary

Negative controls (NC)

NC wells are reactions where no sample input is provided (i.e., buffer only). These are used to assess Limit of Detection (LOD) for each target assay and to assess run quality (e.g., background levels).

NC summary

Negative controls outlier table

The following values for NC wells and targets were identified as outliers and excluded from LOD calculations.

AQ sample controls (AQSC)

AQSC samples are pooled plasma controls from an independent pooled plasma source that is different from CALs. AQSCs are used for calculating intra- and inter-plate coefficient of variation (CV). They are used to obtain an unbiased measure of inter-plate CV when CAL normalization is used.

Note: %CV in this table does not exclude values below LOD. See Coefficient of variation section for %CV (before and after normalization) that excludes values below LOD.

AQSC summary

Heatmaps

Heatmaps show the percent relative to plate median for log2(total counts) and the specified internal control(s).

Plate_01

Quality control

Plate QC

Plate-specific QC criteria:

  • IC CV - Coefficient of variation of internal control Parseable Matching reads across all wells (Maximum threshold = 25%)
  • CAL CV - Coefficient of variation of Parseable Matching reads across all IPCs (Maximum threshold = 25%)
  • CAL Target CV - Median coefficient of variation of all IPC targets (Maximum threshold = 10%)
  • Run Detectability - Percentage of targets that are detectable (target is considered detectable if > 50% of samples are above LOD) (Minimum threshold = 90%)
  • Reads - Minimum number of Parseable Matching reads for the run (Minimum threshold = 100,000,000)
  • AQSC CV - Coefficient of variation of Parseable Matching reads across all AQSCs (Maximum threshold = 25%)
  • AQSC Target CV - Median of coefficient of variation of normalized reads of all AQSC targets (Maximum threshold = 10%)
  • Target Warning - Percentage of Targets with Target QC warnings (Maximum threshold = 10%)
  • AQSC Sample Warning - Number of AQSC samples with Sample QC warnings (Maximum threshold = 0)
  • CAL Sample Warning - Number of CAL samples with Sample QC warnings (Maximum threshold = 0)

QC criteria are assessed either on unnormalized (raw) or CAL-normalized (CAL) data.

Number of warnings: 9

Combined Plate QC Summary

Sample QC

Sample QC summary tables

Sample-specific QC criteria:

  • Detectability - Percentage of targets with reads above the Limit of Detection (LOD) (Minimum threshold = plasma-90%, serum-90%, csf-70%, other-0%)
  • IC Reads - Number of Internal Control (IC) reads within a sample (Minimum threshold = 1,000)
  • Reads - Number of reads within a sample (Minimum threshold = 500,000)
  • IC Median - Sample IC reads relative to the median (Within +/-40% of the plate median)
  • Detectability - Percentage of targets with reads above the Limit of Detection (LOD) (Minimum threshold = plasma-90%, serum-90%, csf-70%, urine-65%, cell_culture-30%, nhp_plasma-55%, nhp_csf-35%, dried_blood_spot-75%, control-90%, other-0%)

Number (%) of samples that have warnings for each QC criterion (excludes CAL, AQSC & NC samples)

Wells with warnings: Detectability

Wells with warnings: IC Reads

Wells with warnings: Reads

Wells with warnings: IC Median

Sample QC plots

Target QC

Target QC summary tables

Target-specific QC criteria:

  • Accuracy - Experimental vs Theoretical average AQSC concentration accuracy (Within +/-30% of theoretical, calculated on AQ targets)
  • CV - Target-specific AQSC coefficient of variation (Maximum threshold = 30%, calculated on AQ targets)

Number of Targets with warnings: 227

Number (%) of targets that have warnings for each QC criterion

Accuracy

CV

Target QC plots

Top % Reads

Top 10 targets

For top 10 targets by % reads on each plate, the table shows target name, % of grand total plate reads, and total target reads. Note that all targets are considered including any excluded or IC targets.

Top 10 targets by % reads

Top 10 samples

For top 10 samples by % reads on each plate, the table shows sample name, % of grand total plate reads, and total sample reads. Note that all samples are considered including any excluded or control samples.

Top 10 samples by % reads

Detectability

Target detectability is the percentage of samples that are above the limit of detection for that target. A target is considered “detectable” if it is above limit of detection in greater than 50% of samples. “Overall” detectability is the overall percentage of samples across all plates that are above LOD for a given target. Target detectability reported here excludes CAL, AQSC, & NC samples.

Detectability summary

Plate_01

Target detectability table

Plate_01

Target detectability boxplot

Plot shows target IC-CAL-normalized count distributions relative to LOD. For each target, log2(LOD) was subtracted from log2(count) for IC-normalized data. Reverse-curve quantified targets are denoted by a “*” and are plotted as forward-curve targets for compatibility

Quantifiability

Target quantifiability is the percentage of samples that are within the dynamic range, that is, above the lower limit of quantification (LLOQ) and below the upper limit of quantification (ULOQ) for that target. A target is considered “quantifiable” if at least 50% of samples fall within the dynamic range. “Overall” quantifiability is the overall percentage of samples across all plates that are within the dynamic range for a given target. Target quantifiability reported here excludes CAL, AQSC, & NC samples.

Quantifiability summary

overall

PLASMA

CSF

SERUM

OTHER

Coefficient of variation

Intra-plate CV summary

%CV was calculated for each target (excluding internal controls) for the AQSCs and CALs. Only values above LOD were used to calculate %CV. Some targets may have missing CV due to too many values below LOD.

Intra-plate CV table – unnormalized

AQSC intra-plate %CV -- unnormalized

CAL intra-plate %CV -- unnormalized

Intra-plate CV table – IC-normalized

AQSC intra-plate %CV -- IC-normalized

CAL intra-plate %CV -- IC-normalized

Intra-plate CV boxplots by plate

Intra-plate CV table – AQ

CVs reported here were calculated on the aM units AQ data. Based on 228 AQ targets.

AQSC intra-plate %CV -- AQ

CAL intra-plate %CV -- AQ

Target AQSC intra-plate CV table

Targets with any intra-plate CV > 30% are highlighted in red.

Number of warnings: 6

AQSC intra-plate CV, IC-normalized

AQSC / CAL ratio plot

Plot shows the log2-transformed ratio (left) or the unlogged ratio (right) of the mean IC-normalized counts for AQSC vs CAL.

AQSC / NC ratio plot

Plot shows the log2-transformed ratio of the mean IC-normalized counts for AQSC versus NC. Asterisk next to target name indicates the NC mean was zero for one or more plates, so AQSC / NC ratio could not be calculated for these plates.

Sample boxplots

Sample boxplots show distributions of the log2 counts for each sample, unnormalized and after IC + CAL normalization.

Plate_01

Sample correlation

Plot shows the Pearson correlation between samples. Hierarchical clustering is done using complete linkage.

Plate_01

Sample and target clustering

Hierarchical clustering of both samples and targets was done using a Euclidean distance metric and complete linkage. Data is log2 and IC-CAL normalized; targets are centered and scaled.

Plate_01

Sample PCA